The overall objective of this proposed research is to investigate mammlian vitamin B6 and its regulation. Particular emphasis will be placed on those tissues (liver and erythrocytes) that show most rapid vitamin B6 uptake, metabolism, and release of metabolites into plasma for transport to other tissues. The regulation of the availability of the coenzyme forms of B6 will be investigated at the levels of their enzymatic synthesis and degradation and of their transport. The binding of pyridoxal and pyridoxal-P to specific proteins in liver, erythrocytes and plasma, and the role of these binding proteins in vitamin B6 transport will be investigated. The erythrocyte and plasma binding proteins will be identified, purified, and characterized as to their affinity for the vitamin B6 forms and inhibition by various compounds. The pyridoxal kinase we have purified will be characterized further. Three other enzymes involved in vitamin B6 metabolism will be purified. These are aldehyde oxidase from mammalian liver and pyridoxine-P oxidase and vitamin B6-P phosphatase from human erythrocytes. The kinetic properties of these enzymes and their inhibition or activation by various compounds will be investigated. The possibility of product inhibition, feedback inhibition, and substrate activation of these enzymes will be studied to elucidate their role in control of vitamine B6 metabolism.